L I E) R. A R. Y
OF THE
U N 1VER.SITY
or ILLINOIS
610 .
V.6 _
no \'^
cop. 2
KLMUIL JdIONAGE
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UMIVtv
The Isocortex of Man
By PERCIVAL BAILEY and GERHARDT VON BON IN
Urbana, 1951
UNIVERSITY OF ILLINOIS PRESS
610.
V.6
no. 3
Melanoblasts and Melanocytes
in Fetal Negro Skin
ARNOLD A. ZIMMERMANN, Dr. es Sc, and SAMUEL W. BECKER, JR., M.D.
Departments of Anatomy and Dermatology
College of Medicine, University of Illinois
ILLINOIS MONOGRAPHS IN MEDICAL SCIENCES, Vol. VI, No. 3
^'^^
UNIVERSITY OF ILLINOIS PRESS
URBANA, 1959
\m 2o m^.
u
UQtCHai.
ILLINOIS MONOGRAPHS IN THE MEDICAL SCIENCES
is a general title used to comprehend a series of contributions from
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Melcnwhlasts and Mclauocytcs
111 Fetal Negro Skni
© 1959 BY THE BOAKI) OK TRUSTKKS OK THK I NIVKHSITV OK ILLINOIS.
MANUFACTURED IX THE UNiTEi) STATES OK AMERICA. Board of Editors:
ERNST R. KIRCH, .lOIlN I'. MARBARCIER, SA.MUEL R. M. REYNOLDS, ISAAC
SCHOUR, AND RICHARD .1. WINZLER.
TMK l.mUMd 111' lONCHKSS IIA.-. CAT M.DI^Kll rH\S I'l Kl.UATlO.N AS FOLLOWS:
ZlM MLIIMAN N. .\nNOLI) .\lHKI1T. 1895-
Mil:iiiol)l:isL< :iiul iii('l:iiu)cyt('s in fetal Nf'(!''<) skin |l>yl .Xinold .\. Zilii-
liiiiliianii Miul Saiiiiirl W. Hi'ckcr. .Ir. I'rballa, rni\ iM-sily of lllinoi.* Frpss,
1959.
,59 p. illu>. '.'7 iiii ( llliii(ii> MioM(ii;iai'lis In nualii-al .Ticnrcs, v. 6, no. 3)
1. ( 'liriiiiiaio|ilioii s. 2. I'jiilirviilony. Unman. 3. Ni'Kio racr. L Hockcr,
.S.'iiiiiii'l William. 1921 iiiilil aiitlior.
{^M1,SI.Z5 tUl.77 58-63808 t
I.iliraiy iif ( 'niinri'ss
1- '- o
Contents
I. Introiluc'tii)n 1
II. Litoraturo 4
III. Materials ami Methods 13
IV. Mi'laiKihlasts and Molaiuicytes in tlic Fetal DiTinis 16
V. The Development of Distribution Patterns of Ejiidennal
^lelanoeytcs During the Fetal Perioil 20
VI. Regional Differences in the Frequency Distribution of
Epidermal Melanocytes in the Negro Fetus 26
^'II. Individual Variability in the Population Densities of
Melanocytes During the Fetal Period 29
VIII. Discussion 30
IX. Summary 34
X. Literature Cited 36
Plates 39
I. Introduction
During the past decade, important changes have occurred in the basic
concepts of the origin and functional rehitionships of mannnalian and
human cutaneous pigment cells. Twenty to 30 years ago, the dominating
view was based mainly on Bloch's theory that ordinary basal cells of the
epidermis were the pigment producers under appropriate stimuli, and that
any true pigment cells encountered in the dermis could have attained their
location onlj' by a descent or Abtropjung either in early or later develop-
mental periods.
Pigment formation in man and other mammals thus was placed in a
separate category. The essential biological events were said to be different
from those known to occur in other vertebrates. That theory failed to
account for pigmentary conditions in certain primates and rodents in
which the dermis is heavily pigmented and the overlying epidermis re-
mains practically free of pigment cells. Adherents to Bloch's theses were
inclined or compelled to consider certain normally occurring pigmentary
features, such as the so-called Mongolian spots, either as histological
curiosities or as having developed from the embryonic epiblast (somatic
ectoderm) at a very early period. Xo evidence had ever been given for the
latter supposition.
The newer and most widely accepted views deny that the skin-
ectoderm has any pigment-forming potentiality. It has long been known
that in other vertebrates, especially amphibians and birds, the source ma-
terial for the cutaneous pigment cells resides in the neural crests. These
structures constitute paired strands of cells that develop as neurectodermal
derivatives on the dorsal sides of the closing neural tube, very early in
embryonic life. They are of an evanescent, transitory nature, never exist-
ing through the full length of an embryo, but differentiating into several
types of cells of which the future cutaneous pigment cell is only one. The
potentialities of the neural crest are indeed impressive.
The credit for experimentally proving that in mammals, too, the pig-
ment cells are derived from the neural crest belongs to Rawles ( 1947,
1948). Her ingenious grafting experiments showed conclusively that in
order to produce pigment, the mammalian epidermis or its hair follicles
are wholly dependent on a migratory cell that enters the somatic ectoderm
secondarily. Rawles never actually identified the migratory forms of the
later pigment cells.
Zimmermann and Cornbleet (1948) observed the first potential pig-
This investigation was supported by a research grant (RG 4436, RG 4436C)
from the Division of Research Grants, United States Public Health Service.
2 ZIM.MKKMANN AM) UKCKKll
meiit cells of tlic tVtal Xcfiio opidermis early in the third luoiith. Thej' are
not luorlified basal cells, wliicli hefiin to reveal melanin granules late in the
fit'tli or in the sixth months — lonj^ after the true pigment producers or
melanocytes begin to elaborate melanin. But no one had yet shown that
the prenatal human dermis contains any migratory cell stages of future
melanocytes.
If the derivation of iiigment cells from the neural crest was to hold
true also for man it obviously became a primary, logical, and challenging
task to attempt the identification of their precursor stages in the dermis.
This report deals in part witli the differentiation of melanoblasts in the
dermis, beginning at 10 and 11 fetal weeks, to melanocytes in the dermis
and epidermis. On the basis of extensive material, consisting exclusively
of Xegro fetuses, we were able to study a pigmentary cell type that hith-
erto had not been, demonstrateil to exist throughout the dermis in the
human prenatal period.
Tlie develoiinient of distribution jiatterns and population densities of
pigment-producing cells in the fetal epidermis was also followed in greater
detail and with more reliable methods than had been applied before. Cer-
tain corrections of published data were made. A strictly statistical treat-
ment of the numerical data, however, was not intended. Instead, we
deliberately placed the emphasis on new and detailed evidence for the
concept that iMgment cells invade the epidermis secondarily. We therefore
established their time of arrival at ultimate destinations in various cuta-
neous regions. This naturally led to cell counts. In the early fetal periotl.
the migration and arrival of melanocj'tes at the dermo-epidermal junction
was found to be iiregular. The variability in cell counts of melanoblasts
and innnature melaiK)cytes in the tlermis of the third anil fourth fetal
months reflects their migration in scattered groups or "swarms." This, in
turn, affects the earliest distribution patterns and population densities of
melanocytes in the epidermis. A reliable biometrical evaluation of such
(lata appeared to be imjiossiljle. The pertinent conditions in the most
interesting developmental peiiod were found to be in a highly fluid state
(see legend to Talkie 1 ).
Since a standardized nomenclature for cellular elements concerned
with pigmentation is not yet established, we define our terms: A "mel-
aniiblast" is strictly an einbiyonic type of cell, potentially capable of
proijucing melanin i)ut not containing the fully elal)orate(l pigment.
Melanoblasts exist only in the dermis and are first detectable early in the
third fetal month. Their round or ovoid cell bodies contain granules that
have a specific affinity for reduced silver but cannot be impregnated with
ordinary silver nitrate. This is generally accepted to mean that such cells
contain a form of "premelanin." Melanin is capable of reducing silver
nitrate directly.
MELAXOBLASTS AND MELANOCYTES IN FETAL NEGRO SKIN 3
The melaiioblasts, recognized by us from the tenth to twelfth weeks of
human development, should be considered as the end stages of a chain of
precursor cells that are derived from the neural crest. The earliest migra-
tory forms in that chain are undistinguishable from ordinary mesenchymal
cells. Since the neural crest of man develops in a cephalo-caudal direction
during the second embryonic month, a gap of several weeks remains in the
demonstration of the ultimate origin of pigment-producing cells. That
gap has been filled by experimentation with mammals (Rawles). There
is no reason to doubt that the events are the same in man as they were
proved to be in mouse embryos. Certainly there is no evidence to the
contrary.
Melanoblasts rapidly develop one to three short, stubby extensions,
the precursors of the future denchitic processes. Thereby they begin to
differentiate into melanocytes. Various transitional forms of fusiform and
young stellate cells, containing increasing amounts of premelanin granules,
make it difficult sharply to distinguish between melanoblasts and melano-
cytes. Our definition of the former is definitely more restrictive than that
implied by the original term "melanoblasf as introduced by Ehrmann
(1885).
Whether melanocytes begin to elaborate melanin while still in the
dermis cannot be used as a criterion for a valid definition. In white skin.
for example, the melanin-producing tyrosinase system of melanocytes is
generally or temporarily inhibited, except in the sacral region (Mongolian
spots). In conditions of vitiligo, the suppression of pigmentary activity is
even more pronounced and it is well known that in albinism there is a
total lack of the enzyme, althougli the melanocytes are present. Billing-
ham (1948, 1949) and Billingham and Medawar (1953) designated such
inhibited cells of white skin as "white" or "non-pigmentary" melanocytes.
Obviously, a broad definition of melanocytes must rely on purely morpho-
logical features. Whether immature or fully differentiated, they usually
have fusiform or stellate cell bodies and several processes. Their potential
melanin-producing faculty may or may not become apparent.
All melanocytes are temporarily in the dermis. Some remain there
until birth, or, as in the sacral region, until later childhood. The great
majority, however, become epidermal melanocytes by an active invasion
of the epidermis. In the Xegro. this process begins in the eleventh and
twelfth weeks of intrauterine development. While still in the dermis, the
fusiform or stellate cells may be designated as immature "dermal" melano-
cytes. This working term, of course, is not meant to imply in any way
that such cells are of dermal (mesodermal) origin, any more than the
epidermal melanocytes would represent a modified epidermal cell. The
adjectives "dermal" and "epidermal" are merely used for the sake of
4 ZIMMKHMANN AM) BEIKKH
brevity ami imlicatc U)catioii as one of the major characteristics of the
cells concerned.
II. Literature
Tlic vast literature on prohlcins and concepts of cutaneous pipmenta-
tioii ill man contains relatively few contributions that deal specifically
witii prenatal conditions. We limit our review to studies that concern
(a) tlie liistolojiical iilentification of pijjment cells in the pre- and post-
natal dermis of man; (b) similar comparative data in other mannnals.
particularly in primates; (c) the recognition of prenatal melanocytes in
the human epidermis; dli data nii population densities and gradients of
distril)iiti()n of such cells; and (e) the experimental evidence for the origin
of manunalian melanocytes fiom the neural crest.
(a) Piyiiieiit cells in the pre- and postnatal dermis of man. The
presence of such cells was first established by Baelz (1885), who identified
them as early as the fifth fetal month in the sacro-coccygeal region of a
Japanese fetus. Baelz consideied the macroscopically visible pigmentation
of that area as an important characteristic of the Mongolian race. Hence-
forth these pigmented, bluish areas became known as the so-called Mon-
golian spots. (Irossly. they had been noticed by Japanese medico-popular
writers for some hundred years liefore Baelz's study, and many supersti-
tious notions liad been attached to them. Baelz noted also that similar
pigmented cells had characteristic relationships to hair follicles, in which
they formed a "regular network" innnediately above the pajnlla.
The first mention in modern science of blue sacral skin areas was by
Eschricht (1849 1, who had obtained second-hand information of their
occurrence in a full-term Eskimo fetus.
An important study of pigment cells in the normal corium of Jai)anese
fetuses, newborns, and children was made by tirimm (1805). Grossly,
he found the blue spots in the sacral region of all newborn Japanese and
noticed that the pigmentation in the spots increased during the first post-
natal months and began to faile in the second .year. In the dermis of the
sacral region Grimm detected the earliest pigment cells in the third and
fourth fetal months. In the Mongolian spot of newborns they were large,
plump, or elongated fusiform cells, sometimes provided with several proc-
esses. The ])rocesses often connected with tho.se of similar iieighi)t)ring
cells, and wiiolc "trains" were tiius interconnected, (iiinuu emphasized
the irregular tlistrilnition of these cells, often in dense "swarms," located
in the deeper two-thirds of the dermis, never in the pajiillary zone. Their
length varied between "JO and 50 /i.
Earliest cell forms of llie tliinl and fonrtii fetal months were round or
MELANOBLAST.S AXD MKLAXOCVTES IX FETAL XEGRO SKIX O
oval, coarsely granular. Some measured 13 ," in the long axis and 7 /t in
width. Grimm conjectured that they originate in very young embryos.
He found no evidence for their derivation from connective tissue cells, as
had been postulated, and cautiously left the question of their origin open
for further embryological and comparative studies.
Grimm found that dermal pigment cells did not invade any eiMilermal
derivatives (sudoriferous and sebaceous glands, root sheaths of hair fol-
licles). Although he confirmed Baelz's observation on the arrangement
of pigment cells in hair papillae, he could not prove that they were
ilirectly derived from the dermis. Grimm's study contains no illustrations
of microscopic sections but shows the gross appearance of the blue spots
in Japanese children,
Adachi i lilU3) was the first to identify pigment cells in the dermis of
the sacral region in infants and children of the white race. He thereby
disproved the opinion that blue spots in the sacral region are characteristic
of a specific (Asiatic) race. The development of pigment in the corium of
localized ( sacral) areas was recognized as a normal occurrence during the
later developmental stages of man in general.
Adachi studied sacral skin specimens of 76 white individuals, ranging
from the fetal period to old age. He made freehand sections of alcohol-
fixed material and obtained satisfactory results with unstained prepara-
tions. He could not detect any blue spots in fetuses of Europeans and was
unable to identifj- dermal pigment cells in white fetuses or in white new-
borns. The earliest spots were grossly recognized in one- to three-day-old
white infants and dermal pigment cells were numerous in the sacral re-
gions of a six-month-old infant and of a 20-month-old child. Adachi de-
scribed the pigmented cells in the deeper dermis as fusiform or stellate,
measuring 40-80 /<. in length and 4^10 m in thickness. Some cells attained a
length of 130 m- He considered them as connective tissue elements and
believed that the pigmented cells of the dermis could not reach or pene-
trate the epidermis. He was puzzled by a constantly pigment-free (papil-
lary) zone between the epidermis and the pigmented layer of the dermis.
Adachi distinguished two types of connective tissue pigment cells:
(a) small cells in the upper layer of the dermis, which obviously corre-
spond to the macrophages of modern interpretation, and (b) large cells in
the deeper layer. The latter unquestionably correspond to our melanocytes
of the dermis and represent the crucial elements of his study.
Besides "dermal " pigment cells, Adachi described epidermal "chroma-
tophores" that are similar in most respects to the dendritic cells or epi-
dermal melanocytes. He believed that they were artifacts produced by
the arrangement of intercellular pigment granules. In holding this view,
Adachi evidently was infiuenced by Schwalbe, in whose laboratory he
I) ZIMMCHMANN AND HKCKKH
worked in Strassl)!!!": and who. with I'nna. Cohn. HaM. and Krouicyer.
was one of the protajioiiists of the thi'orv that cpidciinal "chroniatopiiores"
arc not cells at all. Hanvicr. Ilhiinann. i{ichl, and others held, instead.
that the epidermal pifinient cells ("chroniatopiiores") were fixed connec-
tive tissne cells that had penetrated the epidermis and existed in it inde-
jiendently. Khnnann hail introdnced the term ■'melanoblast." refuting
thai it i(|)i(s(iitcd either a modified leucocyte, connective tissue cell, or an
epidermal cell. .Vccordinji to I'".luinann"s orijiinal definition, "melanoblasts"
were dcrixed from the early emhryoiiic mesodermal layer.
.\da(lii refuted the view that llic .Mongolian spot is an atavistic occur-
rence. Since it is found in many colored races and since European children.
too. have pigment cells in the corium. the potential blue spot or Kinder-
fli'ck had ne\'er been lost in tlie exolution of human pigmentary conditions.
Bloch (1901 I, ill a highly siicciilat ivc report unsupported by evidence, had
maintained that "the spot is a sort of rudimentary organ which gives clues
for the skin color of the ancestors of the yellow race and which one may
call a stigma of atavism."
.Vdaclii licM, instead, that liliic spots are of a i-udimeiitary. regressive
character and that this type of ])igmentatioii occurs in all races during a
certain period of development. His comjiarative anatomical findings on
dermal and epidermal ]Mgmentatioii in jirimates are reviewed under a
separate heading.
Kato (I'.K).")) studied .Mongolian s]iots grossly in r)nn .Japanese children
from biith to 1.'^ years, lie gathered statistical data on their localization^
form, size, color, and rate of disappearance. The spots were also studied
histologically. He recognized the earliest pigment cells in the dermis of an
eight-month fetus. In some skin specimens of children he found pigment
cells often adjacent to blood vessels. The cells of a dee]i blue .Mongolian
spot ill a iiiiie-iiioiitli-iild chilli were rdiiiid, oxal. nr tiisifdrm. and of a
brown color. .Many branched cells ap])eai-ed connected with each other,
and though the papillary zone was mostly free of jngment cells. Kato
obser\'e(l tluMii occasionally at the (lerinii-e]iiileiiiial junction. He made
one of the Hist histological studies of a liliie nexus (adult .lapanese) and
identified fusiform, branched, or serpentine cells of brown color in the
ileeper two-thinls of the cutis vera. He considered the characteristic jiig-
meiit cells ill the dermis of the sacral areas of children and of the blue
nexus ill ailiills as iileiitical. (Iriiiiiii had expressed a similar oi>inion
xxithiMil gixiiig histological ex'idence.
Pile gradual disappearance of llie .Mongolian sjiols in later chililhood
was e\])iaiiied by I lie disiiit egi at loll of pigment cells and the absorjition of
|)iginent granules by dermal lymphatics and venules. In the blue nevus
of adults the cells xvere said to remain functional and to i(>tain their
characteristic form.
MELAXUIiLASTS AM) MKLANorVTKS IN KKTAL NKliHO SKIN 7
Bloch (1921) also attempted to fiiul the site and time of the earliest
pigment formation in white fetuses. His material consisted of 12 speci-
mens ranging from the third to the nintli month. Pigmentary elements
within the dermis were encountered in the sacral legion of a fetus of about
five months. Bloch described them as "peculiar cells which are not re-
lated to the normal pigmentation of the epidermis." These dopa-positive
melanocytes in the dermis remained "a puzzle as to their origin and sig-
nificance." They were the only exception to Bloch's conccjit of the local-
ization of pigment production in strictly ei)itlielial cells of ectodermal
origin. Since he detected them only in the sacral area (Mongolian si)ot),
Bloch designated them as Mongolenzellen. Nonetheless, he was aware
that a complete answer could not be expected from his limited material.
El Bahrawy (1922) studied under Bloch and tabulated the published
reports of Mongolian spots according to their geographical and racial dis-
tribution. They were seen macroscopically in newborn and children: 100
per cent in Japanese and other Mongolians, SO per cent in Negroes anfl 2
to 4 per cent in Europeans. In white children without visible sacral spots
the pigment cells in the dermis were pale, scarce, and easily missed. He
made histological observations on sacral skin of 112 European cadavers
(unstainerl. stained sections, and silver impi-egnations accorrling to Biz-
zozzero). Pigment cells in the sacral dermis were first identified in a few
white fetuses of four to fiv(> nn)nths and in all specimens from five fetal
months to nine-year-old children. In specimens from 12 to 82 years of
age, dermal pigment cells were seen in only four, none over 21 years old.
The irregular distribution of these cells was noted in many preparations.
Oval, pear-shaped, fusiform, or irregularly wa-^'y cells measured 5-10 by
30-50 M.
The argentaffin cells of the dermis were shown to be dopa j^ositive and
were recognized, therefore, as true pigment producers or "melanoblasts."
Their normal occurrence in European children was considered as a histo-
logical or racial curiosity. Bahrawy recognized that phylogenetically such
cells might represent "a temporary remnant of generalized pigmented
areas in animals, where they exist throughout life."
In a biopsy sj^ecimen from the forearm of a rhesus monkey, Bahrawy
found numerous fu.siform pigment cells in the deeper layers of the dermis.
These dopa-positive cells were believed to correspond to those of the
Mongolian spot in man. The typical cells of a blue nevus and tlic "Mon-
golian cells." as well as the dermal pigment cells of certain monkeys, were
thought to be of the same nature. This was an important advance in the
direction of a broader concept. Bahrawy speculated, however, that the
cells in question might be of early ectodermal origin and had migrated into
the dermis during embryonic life. This represents an attempt to support
his teacher's theory.
I
8 ZIMMKIIMANN AMI BKCKKH
Isliikawa (1!)24) searched for the characteristic piKiiicnt cells of the
sacral derinis in 32 Japanese fetuses. The earliest ones were found in two
sppcinions (of a ^roup of ton fotusos) of tlic early part of the third month.
Tlicy wi'ic rt)un(l oi' o\;d cells, nica.'^uiinf; lO-lo n. rarely 23 /i. and showinjj
lui processes, (leneially lliey were hifihly scattered and contained yellow-
ish-brown pigniont fj''i"iilpsi. Similar cells were identified in the deeper
layers of the sacral corinm in four of eijiht fetuses of the fourth month.
Tlieie were numerous i)ijimeiit {'ells in the .Monjiolian spot areas of fetuses
ranginii fidiii the lit'tli to tlie tenth iiidiith. In specimens older than six
months they wei'e mostly spindh'-shaju'd and measured 4-10 by 30-80 f^.
Ito (l!).")^) reported on dermal piunient cells of the Monjiolian spot.
tlie l)lue nevus, and the nevus fusco-coeruleus (of Ota). He stated that they
were i)roiUK'e(l l)y "mesenchymal melanohlasts.'" Definite affinities with
the nervous system ("sugfjestiuii eiido- or iiei'inevnium"' ) were postulated.
Tins concept is reminiscent of Weidenreich's obsolete theory. Ito believed
that during its gradual disappearance the Mongolian sjiot becomes es-
tranged from the nerxous system, whereas the more localized conditions
of the blue nevus and nevus of Ota retain their nervous coimections for
life. In his studies of Mongolian spots in Japanese fetuses of varying ages.
Ito reported results almost identical witli those obtained by Kato and
Ishikawa. As late as 1957 (second report on melanin studies) Ito stiU
maintained that melanogenous dendritic cells were derived from peripheral
nerve fibers.
Barry (1952) was the first to report dermal pigment cells in other than
the sacral region of man. Working in a French laboratory at Hanoi. Indo-
china, he studied fetuses of the yellow race and some scalji specimens of
newborn and adult individuals. The earliest dermal melanocytes were
identified in silver-impregnated head sections of a 5 cm fetus. We calcu-
lated its age to be almost three months. The cells containcMl jiremelanin:
some had short processes and were located in the reticulopajnllary zone
with their long axis lying parallel to the basement membrane. Some ap-
peared to lie migrating toward th(> e])idei'inis. witli processes extending to
the baseuKMit membrane. Tliere were as yet no demlritic cells in the
epidermis. (French authors persistently designate the latter cell type as
"Langerhans cells.") Scalp sections of a fetus of 3.5 months (8.5 cm
C.R.L.) showed a few dendritic cells in the epidermis and some dermal
melanocytes. In silver-stained scalp sections of a fetus of 4.8 months
( 14.5 cm C.H.L.) there were numerous e)M(lermal dendritic cells and vari-
ous forms of dernial nielano('\tes. '{"here \\v\v also dendritic cells and
dermal melanocytes in the lumbodorsal-gluteal region. The illustrations
of the latter cells resemble our own photomicrographs of similar cell stages
in younger Negro fetuses. At five months tlie dendritic melanocytes of the
MELAXOBLASTS AND MELANOCYTES IX FETAL XEfiRO SKIX 9
epidermis were numerous in the lumbar legiou but the basal cells con-
tained no pigment granules detectable with silver. There were many
branched pigment cells in the dermis of the scalp, often associated with
blood vessels. A similar perivascular arrangement was noted in the scalp
of a newborn. The basal cells now contained fine pigment granules, seen
both in unstained and silver-treated sections. Xo dermal melanocytes
were found in the adult scalp, but the basal cells were now crowded with
melanin granules capable of reducing silver nitrate.
Barry concluded that the dermal melanocytes are the forerunners of
the dendritic "Langerhans cells." At first they are without processes and
contain only premelanin. Later, branched forms appear, some of which he
presumed to become the "dendritic melanoblasts" of hair follicles. In all
races pigment granules appear in the basal cells of the epidermis long
after the "Langerhans cells" have reachefl their destination. Barry noted
that the melanocytes disappear in the dermis when the population of
dendritic cells within the epidermis attains a certain density. This is the
first suggestion of a puzzling "barrier" effect that seems to regulate the
number of invading epidermal melanocytes per unit skin area.
Barry believed that the dermal melanocytes were derived "probably
from the ectomesoderm." without specifically mentioning the neural crest
and without being aware of Rawles's work. He postulated that the pig-
ment cells of the Mongolian spot, blue nevi, hair follicles, and the dendritic
cells of the epidermis originate from the same source. He strongly opposed
the theory of Bloch and adduced valid evidence for his plea that it should
be abandoned.
(b) Comparative anatomical data on dermal and epidermal pigmenta-
tion in primates and other tnammals. Adachi (1903) made important con-
tributions to our knowledge of jjigmentary conditions in the skin of apes
and monkeys. In primates, cutaneous pigmentation exists both in the
epidermis and in the dermis (orang, chimpanzee), sometimes only in the
epidermis (gibbon, spider monkey), or only in the dermis (baboon, bar-
bary ape). There were variable amounts of melanin in either layer and
the type of cutaneous pigmentation was unrelated to the animal's syste-
matic position. In some anthropoid apes (orang and chimpanzee) Adachi
observed great numbers of pigment cells in the dermis of practically all
body regions. They were spindle- or star-shaped cells, measuring 80-150
by 5-10 /i. Similar cells were characteristic for the dermis of various
species of Macacus (rhesus monkeys), which have a bluish skin and little
epidermal pigmentation. In general the extensor side of the extremities
was richer in pigmentation, in either epidermis or dermis, than the flexor
side. In the chimpanzee the dermal pigment cells in the extremities often
were adjacent to blood vessels.
It) ZIMMKUMAN.N AM) Hi:< KKIi
Since ill adult man the dcjirco of opidcinial pifinipiitaf ion \'ari('s {jroatly
wilii ract', ami the coiium is tree of laifio pijinient cells, Adaclii considered
"Homo" as an independent piffmeiitary type.
Miescher (1922) identified similar pi^nK'nt cills in certain dermal
areas of the mouse. They were iiuiikmous around the \il)rissae. where
they formed a collarlike network at the upper end of the follicles. Blood
vessels were also aecompaiiicd by dermal pigment cells, wliich Miescher
described as threadlike, elongatetl, sometimes branched or star-shaped.
Their dopa reaction was positive but variable. He therefore recognized
them as "autochthonous pigment-producers" and likened them to the
pigment cells of the chorioid of the eye. Assuming that the pigment cells
in the cutis vera of the mouse, apes, and some monkeys, and those of the
chorioid and of the Alongolian spot were of mesodermal origin, Miescher
acknowledged that the principle of an ectodermal pigment formation could
no longer be maintained or, at least, appeared questionable,
Daniieel and Cleffnianu (1954) showed that various species of rodents
liave pigmentary conditions similar to those in apes and monkeys: the
dermis, the epidermis, or the hairs may be the only pigmented structures.
They studied embryos and skin specimens of newborn mice. rats, and
rabliits. The earliest dermal melanoblasts were identified in mouse em-
bryos of 14 to 15 days. The cells were relatively scarce and located near
the dermo-hypodermal junction. None were directly beneath the epi-
dermis. This had been construed to mean that there is no relationship
between dermal and epidermal pigmentary elements. Danneel antl C'let^"-
maiin believed, however, tliat dermal melanoblasts rapidly ascend and
])cnetrate the epidermis. They observed that in the ear of mice and rats
such migrations occur relatively late, one to three days after birth. In the
ear the tlermis retains some pigment cells throughout, whereas in other
body regions they tlisappear. On the backs of rabbits only hair follicles
contain melanocytes. Danneel and Cleffniaiiii never .^aw a ilirect migration
of dermal melanolilasts into developing iiair follicles. The follicles receive
their pigment cells from the epidermis during development by migration
along the outer root sheath. The migratory process of melanocytes stops
soon after birth, and tlic increase in iiuinber of cells dccurs liy rejieated cell
di\isioii in thi' papillae^. Tliese investigators were aware of Rawles's ex-
l)erimeiital evidence and agree(l witli tlie view that all pigment-producing
cells of vertebrates are derived froni ihe neural crest.
Weissenfels (195(5) contributed interesting details on the earliest
jihases of melaiiogenesis in embryos of .Japanese "silky" fowl. The epi-
dciinis and featiiers of aduil "silkies" are devoid of i)igiiieiit, l)ut the
uiMlcrlyiiig tissues coiilain iiunicrous pigiiuMil-pro(hiciiig cells. Without
gi\iiig (liicct cxidi'iicc. the aiillior staled tlial in carlx' ciiilirvonic stages
MELAXOBLASTS AXD MELANOCYTES IX FETAL XEGRO SKIX 11
"spindle-shaped cells migrate from the neural crest into almost all body
regions. " In tissue cultures he observed the origin of premelanin granules
within specific cytoplasmic centers of melanoblasts. Granules were pro-
duced periodically in waves. The centers were not identical with the Golgi
apparatus. Preparatoiy to mitosis the melanoblasts became more spherical.
Some processes, crowded with granules, remained connected by slender
plasma bridges. After the nuclear events of mitosis, one of the daughter
nuclei usually migrated into one of the retained cell processes. No flow of
pigment granules could be obser\ed from mother to daughter melanoblasts.
The latter, instead, soon began to produce their own premelanin granules
from newly arising cell centers. They often formed budlike evaginations
of the cell contour from which the demhitic processes arose. These events
were observed both in vivo and in vitro, with the phase contrast and the
electron microscope.
(c) Data on prenatal melanocytes in the human epidermis. Pigment-
producing cells in the epidermis of Negroes before birth were discovered
relatively late in the history of pigment research. The prevailing opinion
had been that Negroes were born white and that pigment appeared only
during the first few postnatal days, especially at the nail folds, areola of
the nipples, and the external genitalia. The concept was based in part on
the dicta of renowned histological authorities, e.g., Kolliker, Unna, and in
part on careless examination of the hyperaemic, pink skin of the newborn.
Morison (1889) was one of the first to state that Negro children were
born with some cutaneous pigment. In skin sections from the arm of an
eight-month Negro fetus he observed pigment in the deepest layer of the
epidermis.
Thomson (1891) identified pigment, in unstained sections of the scalp,
in a five-month Negro fetus. He also observed the curved character of hair
follicles as well as "interlacing pigment cells" in the hair bulbs.
Grimm (1895) had seen small amounts of pigment in the rete mal-
pighii of Japanese newborn. Adachi (1903) corroborated this and further
observed that newborn whites often had a lightly pigmented epidermis.
His concept of the epidermal "chromatophore" as a non-cellular structure
has been discussed above.
Bloch (1921) obtained the earliest dopa reactions in the skin of a five-
month white fetus. No true pigment was detectable in either epidermis or
dermis, but certain melanoblasteiiartige Zellen within the basal layer re-
vealed a gray-brown hue. In another specimen of the same age, the matrix
of hair bulbs contained "a few cells with processes that looked like 'melano-
blasts' and gave a weak, positive dopa reaction." Neither the papillae nor
the hair shafts contained any pigment as yet. Fully formed melanin was
first identified in a few hair Inilbs of the fifth fetal niontli and in the
12 ZIMMKKMANN AND IIKIKKH
cpidorinis i)roper of sppcimcns of the sixth to seventh iiioiith. The dopa-
positive cells within the basal layer were descritjed as typical "inelaiio-
blasts" (in the sense of lihrniann's definition): irregular, star-shaped cell
ixxlies with l)ranch('(i inoccsscs. Bloch compared them with "ganglion cells
of the brain cortex." He stre.-Jsed again that all pigment was produced by
cells of ectodermal origin (basal layer of the epidermis and hair matrix).
Ziinmciiiiaiin timl ( '(iriil)leet (1!)4S) recognized dendritic melanocytes
within tiie ("jjidermis of Negroes early in the third fetal month. They ob-
taineil jiositive dopa reactions, exclusively in tliese cells, from the fourth
fetal month on. The transfer of melanin granules from melanocytes to
neighboring epithelial cells was first noticed late in the fifth fetal month.
The papillae of lanugo hairs were seen to contain dendritic melanocytes
whose ])rocesses extended directly into the ba.«e of the hair shafts. The
melanizatioii of hairs occurred independently of the so-called epithelial
matrix, which itself became pigmented later on. The ''interlacing pigment
cells" noticed in' Tliomson evidently correspond to the dendritic melano-
cytes, which lie between ordinary matrix cells of the hair bulb.
These conditions were further studied by Zinnnermann (1954). who
also attempted the first evaluation of the numerical density of melanocytes
in the fetal epidermis of Negroes. The intercellular distances of 200 con-
secutively encountered melanocytes were measured in serial sections.
These distances decreased by api^roxiniatcly one-iialf Ijetween the fourth
and the fifth months. It was concluded tiiat the munber of tlendritic cells
had doublet! during that interval.
Becker and Zimmermaim (1955) canied tiuit numerical analysis fur-
ther, ("ell counts weie made in spreads of separated epidermis. In the
newborn Negri) they found apjiroximately 1.000 doiia-iK)sitive melanocytes
per nun'-. This figure compares favorably with similar counts made by
Szabo (1954) in adult white skin. Clold chloiide impregnations revealed
the earliest dentritic melanocytes in the epitlermis of white fetuses at six
luonths. In Negro fetuses of the third month the first mature melanin
gianules were identified in melanocytes of ihe eyelids, the exteriud audi-
tory meatus, and s])ecific ai'eas of tlie oi'al nuicosa.
Ilu. Staricco, I'inkus, and Ft)snaugh (1957) made observations on
melanocytes of the prepuce of white and Negro infants. Their illustrations
of cells in tissue cultures show many types resemtiling those described in
the present study. They found thai "the iclal i\-el\- small ]iiginent cells in
I he outgrowth of normal skin explant resemble the early melanoblasts of
fetal life rejiorted i)y Zinunermann and ('orni)leet. ' Young pigment cells
were recognized in the cultures as bipolar or stellate cells. .\s they maturetl
they became strongly dopa positive and showed riclier dendritic ramifica-
tions. There were no transitional forms ijetwceii onhnarx' ei)ithelial cells
antl melanocytes. Kach cell type gave rise to daughter cells of its own kind.
MELAXOBLASTS AND MELANOCYTES IN FETAL NEGRO SKIN 13
(d) Evidence of gradients in the devclop)iient of cutaneous piynienta-
tion. Although the migration of nielanoblasts from the neural tube to
various body regions had been well established for certain vertebrates,
there is only fragmentary information on the rate at which they arrive at
ultimate destinations. Hopkins-Fox (1041)) first determined such a
schedule in embryos of barred Plymouth Rock chicks. The migratory
nielanoblasts could not be identified with certainty in histological prepa-
rations. The evidence, therefore, was based on the end results of grafting
experiments. Of more than 2.000 transplants, about 1,000 were successful.
The earliest migration of nielanoblasts from the neural tube occurred at
the level of the mesencephalon, in chick embryos of 8 to 10 somites. In
embryos with more than 27 somites "the epidermis overlying all somites
tended to yield nielanoblasts upon transplantation." Roughly, an antero-
posterior sequence in the migration of nielanoblasts was revealed. In the
limb-buds there was a proximo-distal gradient as well as a dorso-ventral
migration. The migration was not limited to the epidermis, but nielano-
blasts also reached visceral structures along blood vessels (coelomic lining,
mesorectum, testes).
(e) Experimental evidence for the origin of mammalian melanocytes
from the neural crest. The fundamental woik by Rawles (1947, 1948) was
briefly referred to in the introduction. The pigment-forming potency of
various body regions of mouse embryos of a black strain was tested by
transplanting them into the coeloni of white Leghorn (albino) chick
embryos. Only tissue grafts that contained presumptive or definitely
identified neural crest were able to differentiate melanocytes. A medio-
lateral spread in that pigment-forming capacity occurred, first at cranial
and later at caudal levels. By several hundred grafting experiments Rawles
proved conclusively that the somatic ectoderm or its hair follicles are
incapable of producing their own melanin. In mammals, too, that faculty
belongs exclusively to nielanoblasts and melanocytes, which are derived
from the neural crest.
Such experimental proof is not feasible in man. A search for the
earliest phases of melanogenesis by histological means, therefore, appears
to be the only possible approach. The results of our own endeavors are
presented in the following pages.
III. Materials and Methods
One hundred and seven Negro fetuses were collected through the
courteous co-operation of various hospitals and institutions: the Depart-
ment of Obstetrics and Gynecology of the Illinois Research and Educa-
tional Hospitals (Dr. W. F. Mengert), the Department of Pathology of
14 /.IMMKU.MANN ANt) Hl;< KKK
the University of Illinois. College of Medicine (Dr. C A. Krakower);
Cook County Hospital. Departnient of I'atludogy. Chicago (Dr. P. H.
Szanto): tiie Carnegie Institution of Washington. Department of Embry-
ology, Baltimore (Dr. C Corner); Tulanc liiiversity. New Orleans. De-
jiartment of .\natomy (Dr. II. Cununins) and tlie Department of Medicine
{Dv. \. l)erl)es); Los Angeles County General Hospital. DeiJartment of
Surgical Pathology (Dr. W. Hullock): the Chicago Maternity Center (Dr.
Beatrice Tucker), and tlic .Molinc City Hospital. Illinois (Dr. X. T.
Braatelien).
We extend our sincere thanks to all those wiio made this material
available to us.
Al)out (K) specimens were particularly well preservetl. All were
formalin-fixed. Detailed records were kept concerning the findings on
melanocytes in the dermis and e])idermis. Other specimens were dis-
carded liecause tliey were too yoiuig. sIiowcmI varicMis flegrees of maceration,
or were otherwise inadequate for our study.
Age determinations were made from careful measurements of crown-
rump length and by applying the formulae of Scammon and Calkins
(1929):
C.R. Length = .(i(j C.H. Length (cm)-|-.5 cm
and
. fC.H. cm ,,.-,. I " , -,
Age= j — ^g -|-L2o| +./4
These rules give menstrual age in lunar months of 2S days.
The ages of several yoimg fetuses obtained from the Carnegie Institu-
tion of Washington had been established according to the rigorous criteria
of that laboratory. Our own calculations coincided with theirs. We were
able to obtain only skin specimens from several older fetuses (twenty-
sixth to twenty-eighth week) and newborn Negroes from Cook County
Hospital. Chicago. We accepted tlie ages given by the hospital's Depart-
ment of Pathology.
Where\('r feasil)le we made skin shavings from 21 selected body
regions of each fetus: two from scalji and cheek, seven areas from the
trunk, six dorsal and volar areas of the ujiper limb including the palm, and
six posterior and anterior regions of the lower limb, including the sole. In
fetuses of the thin! and fourtli months, the fidl thickness of the delicate
skin could be usc(|. in older t'cluses, two horizontal slices usually were
made, one containing tiie e!)idermis with the upper portion of the dermis
and another one consisting of the tleeper dermal layer. By stretching the
skin areas of the sjiecimens it was jiossible to make the slices by hand.
nicii'ly using a la/or blade, .\fter staining, the jireparations were mounted
as spreads, alternately willi tlie dermal or tiie epidermal surfaces upper-
MELAXOBLASTS AND MELAXOt'YTES IN FETAL XE(iRO SKIX 15
most. Surface examinations of such spreads clearly revealed the number
and manner of arrangement of melanocytes. Population densities per mm-
could be determined more accurately than is possible in sectioned material.
Our extensive slide collection was prepared by Dr. Hans J. Knoblich.
We are glad to acknowledge our indebtedness for his fine technical assist-
ance and faithful co-operation.
StainiiH/ Tfcliiiiquc. The best results were gained by modifying
Masson's impregnation method of ammoniacal (reduced) silver nitrate.
After thoroughly washing the formalin-fixed skin slices in distilled water,
we placed them in a freshly prepared and filtered solution of 10 per cent
ammoniated siher nitrate. Masson's original procedure required 6-8
hours impregnation time at room temperature. We incubated our prepa-
rations at 55' C from 10 to 30 minutes. Frequent checking of the speci-
mens (in distilled water, under the microscope) prevented overstaining.
Usually a sepia-brown tint of the skin slices indicated that the incubation
could be terminated. Even in such non-cleared preparations the epidermal
melanocytes could be readily detected by low power examinations. The
quality of the fixation appeared to affect the staining time.
Adequately impregnated skin spreads, usually about 1 cnr in size,
were then treated with a (3 per cent solution of sodium hyposulfite. Gold
toning was omitted. The preparations were then dehydrated in an alcohol
series, placed in xylol, and mounted in "Permount" as spreads.
The study of vertical skin sections was accessory. Sections were used
mainly for the accurate determination of the depth at which melanoblasts
and incompletely differentiated melanocytes were found in the dermis.
These sections also were impregnated with ammoniated silver nitrate and
incubated at 55° C. The stain was no better, but much faster than that
produced by the original ]\Iasson technique. After gold shading, the prep-
arations were finished routinely.
Cell Counts. Cell counts were made in over 500 microscopic fields of
epidermal anrl dermal melanocytes. All counts were obtained by means
of camera lucida projections. The standard fielcl measured (0.33 mm)-^
1/9 mnr. Each observed melanocyte was traced, and after the field was
completely surveyed the sketched-in cells were counted. Their number
was then multiplied by nine to obtain an estimate of their population
density per mm". Obviously, any error in counting was also multiplied by
nine. However, checks were niade by tracing and counting the cells of one
particular field on ten different plots. At other times the cell counts in a
given field were made by dift'erent observers. In either case the errors
were negligible, primarily because the silver impregnations were of high
quality and the melanocytes easily identified. A comparison of cell counts
in any two fields appeared justified and reliable.
16 ZIMMKHMANN" AMI HKCKKH
The senior autlmr is responsil)k' fur practically all cell counts and for
the analj'sis of the data.
The photoniicrojiraphs wore inarlc by Mr. Lawroncp Toricllo. Illiistra-
tioii Studios, I'liiversity of Illinois. His skillful work is greatlj- appreciated.
IV. Melanoblasts and Melanocytes in the Fetal Dermis
\\ c si'arclu'il for i)rccursor stages of melanocytes in many body regions
of n-l selected specimens. Many of these fetuses were also used for the
study of epidermal melanocytes reported on in succeeding sections.
Questions of nomenclature were discussed in the introduction. We are
using the term "melanoblast" for an embryonic type of cell, potentially
able to produce melanin. A "dermal" melanocyte is a more highly differ-
entiated cell, fusiform or stellate in shape, containing prenielanin or
melanin, and is located in the dermis. It is also designated as an immature
melanocyte. An "epidermal melanocyte" is the fully differentiated,
dendritic type of cell, also elaborating melanin aiul located exclusively in
the epidermis.
"Dermal" melanocytes become visible in uiisfaiued fetal skin spreads
as early as the fourth month (figure 7), Their granules are undistinguish-
able from melanin and have affinity for reduced silver nitrate (Masson).
Doi)a reactions were not feasible in our formalin-fixpd material.
Tenth and Eleventh Weeks of Fetal Dei'elopment. The earliest
melanoblasts were identified in skin specimens of ten Xegro fetuses of this
developmental period. Their crown-rump length ranged from 3.4 to 4.5
cm. Preparations from various body regions were obtained by stripping
small pieces of the delicate skin. In eight fetuses of this group the melano-
blasts were found only in the scalp, in others also in the nape and in the
saci'al r(>gion. 'i'hey wei'e identified as round cells oi from S to 12 /i di-
ameter. Their eccentric nucleus usually contained one or two nucleoli.
Fine argentaffin granules were disseminated throughout the cytoplasm.
Due to the spherical shape of these cells, they appearcil to be more densely
arranged at the jierijilieiy.
The relatively large melanoblasts were widely tlispersed in the con-
nective tissue. .\t that stage of development a true dermis cannot be
distinguished from tiie hypoderniis. Cell counts were not made because of
the scarcity and wide scatter of these elements. The earliest precursors of
future ])igment cells were also recognized in strijiped skin pieces simply
mounted in water and examined under the microscope. Such cells contain
refractile granules which make tiiem readily iilentifial)le. The size of the
refractile granules corresjionded to that of the argentaffin granules seen
after im]ir(^gnation with reduced silver nitrate.
MELAXOBLASTS AND MELANOCYTES IX FETAL XEGRO SKIX 17
Changes in form of the round nielanoblasts occur ah-eady during the
tenth week of development. Some were ovoid, assuming the shape of fall-
ing drops or of lemons with two small projections at each pole. Gradually
more and more fusiform cell types appeared, often of 20 n length. The
argentaffin granules tended to accumulate in the tips of the cell processes,
giving the impression of active "growth points," Some ovoid cell bodies
had two processes at one pole, foreshadowing a tripochil arrangement of
future dendritic processes.
Common to all forms were the argentaffin granules of very fine, even
size. Their presence and the continuous series of cell shapes fi'om round
to stellate forms were the cytomorphogenic features indicating a single
lineage. In scalp spreads of the eleventh week some spindle-shaped cells
measured between 30 and 45 m as compared with an average diameter of
only 15 /i of the overlying epidermal cells. In general, the differentiating
melanocytes of the dermis were conspicuously larger than fibroblasts or
fibrocytes of their surroundings.
During the tenth and eleventh weeks of development, melanocytes
in the epidermis were rare. Round nielanoblasts and immature melano-
cytes in the fetal dermis, therefore, precede the first appearance of epi-
dermal melanocytes (in numbers) by about two weeks.
Twelfth Week. In nine fetuses of this period, nielanoblasts and im-
mature melanocytes were identified in many body regions. We consider
this as an indication of the rapid migration or arrival of pigmentary
precursor cells from their presumptive source in the neural crest. The
crown-rump length of the specimens varied from 5.0 to 6.7 cm. Since
the skin was still very delicate, full-thickness strips could be used. ]\Ielano-
blasts and transitional forms of "dermal" melanocytes were observed in
the scalp, cheek, nape, interscapular, and sacral regions, in dorsal areas of
the forearm, of hand and foot, anterior aspect of the leg, and even in the
palm and sole.
The first cell counts of dermal nielanoblasts and of incompletely
differentiated melanocytes were feasible. The distribution of "dermal"
melanocytes, however, was not uniform through a given field. They ap-
peared in groups or "streams" of considerable accumulations. Often the
long axes of the spindle-shaped cells were parallel to each other, indicating,
perhaps, a directional flow through the connective tissue spaces.
The cell counts given in Table 1 cannot be taken as an absolute meas-
ure of population densities. They merely indicate the relative frequencies
with which nielanoblasts and "dermal" melanocytes were encountered at
this early age. The table contains the calculated number per mnr of both
"derniar' and epidermal melanocytes. The great variability in the number
of epidermal pigment-producing cells is due to their irregular distribution
pattern at this early age.
18 ZIM.MKK.MANN AM) HKIKKU
Table 1. Cell Counts of Melanohlast.s anu Kauly
Epidermal Melanocytes in the Twelfth Week
l?(uly regions
Mclanohlasts and "dermal
melanocytes per mm-
Epidormal melanocytes
per mni-
in corresponding areas
Scalp
03,
182,
198,
387
' — .
189
Interscapular
198
90, 144,
180, 315
Forearm, dorsum
4o,
1G2
171
Hand, dorsum
144,
2.J2
90,
13.j
Palm
198
234,
279, 351
Leg, ant. region
126
90,
36
Foot, dorsum
378
18
Sole
162
63, 72,
153, 243
The wide range of cell counts in a given region is due partly to the migration and
arrival of melanocytes in irregularly scattered groups or "swarms." Stabilized and more
typical distrihution iiattcnis of mclandcytes become established in the fourth and fifth
fetal months.
Numerous mieronietric measurements were made with high power
magnification. The spherical melanubhists measured from S to 10 m.
teardrop or lemon-shaped forms from 12 to 16 ti-. Cells with stubby
processes varied between 20 and 25 n. Spindle-shaped forms of immature
melanocytes measured from 30 to 45 n in length.
Figures 3 to (i siiow high power pliotomicrogiaphs of a roinid melano-
blast and of early tonus of "deiinal" melanocytes (twelfth week). Obvi-
ously, sharp focussing of these cells in the dermis of skin spreads is more
(Ufficult than in sections.
Thirteenth and Fourteenth \\'( el.s. The six fetuses of this fetal period
varied in crown-rump length from 7.0 to 0.5 cm. Melanoblasts and
"dermal" melanocytes now were present also in the pectoral region and on
the anterior aspects of arm and thigh. Figure 0 illustrates early forms of
melanoc\'tes in tlie dennis of a lumbosacral spread.
Cell counls varied gencrall.N' helween 200 and 4(11) jier mm-'. In one
fetus, however, the counts wcvv nuicli higher in the sacral region and in
the dorsum of hand and foot. Specifically, the comits were ()S4 and 1.305
for the sacral region, 003 and 1.101 for the dorsmn of the hand, and 855 in
the ilnrsuiii (it tlie foot, ('nunts of tiic sanu' cell types in the palm and
sole were the lowest for tlmt specimen: lOS and 201, respectively.
During the thirteenth week yoimg ei)idermal melanocytes appear in
many skin regions. Sudi juvenile forms usually are slender, fusiform cells
with long |)riniary dendritic processes. They are easily recognized in a
liiglici' focal plane lli:in llial of the "dernial" inelan(ie>'tes. The latter also
MELANOBLASTS AND MELANOCYTES IN FETAL NEGRO SKIN 19
teiul to be spindle-shaped with cell processes just beginning to form. Fusi-
form "dermal" melanocytes from the dorsum of the foot are shown in
figure 8.
Fifteenth and Sixteenth Weeks. The seven fetuses of this develop-
mental period, terminating the fourth month of pregnancy, varied in
crown-rump length between 9.5 and 11.7 cm. The population density of
"dermal" melanocytes, in general, was higher than in the preceding weeks,
although there still was considerable variation. This may have been due
partly to technical procedures. If surface shavings are too thin the prep-
arations may reveal only part of the population of dermal melanocytes.
Cutaneous areas from the scalp, and dorsum of the hand and foot, some-
times contained 500 to 700 melanoblasts and "dermal" melanocytes per
mm-. Spindle-shaped forms usually were predominant, although in one
fetus of 16 weeks many round melanoblasts were typical of various regions,
particularly in the dorsum of hand and foot.
During this developmental period, "dermal" as well as epidermal
melanocytes become visible in u)}stai})ed preparations. Figure 7 shows
spindle-shaped cells in the dermis of an unstained scalp spread (fifteenth
week.) The presence of true melanin in those cells was proved by impreg-
nations with ordinary silver nitrate (Bizzozzero's method). Relatively
young forms of "dermal" melanocytes from the dorsum of the hand are
shown in figure 10.
Seventeentli to Twentieth Weeks. Split-skin preparations were made
of 15 fetuses of the fifth month of pregnancy. Their crown-rump length
varied between 12.5 and 16.5 cm.
Cell counts of "dermal" melanocytes remained high in most regions.
There were over 1000/mm- of such cells in the sacral region (Mongolian
spot) of two specimens (figure 11). Counts of epidermal and "dermal"
melanocytes of a preparation usually were lower by one-half or two-thirds
for the latter type of cells, except in the sacral region. Cell counts in
preparations impregnated with reduced silver were consistently higher
than those in unstained skin spreads. This agrees with the generally ac-
cepted view that ammoniacal silver nitrate reveals precursor stages as well
as fully elaborated melanin granules. In unstained preparations presum-
ably only the latter are visible.
In some fetuses of this period, the spindlc-shapetl melanocytes in the
dermis were the most characteristic cell type. They had become longer
and measured from 40 to SO /i. In other specimens the majoiity of the
"dermal" melanocytes were of the round or ovoid form. They may have
been "young arrivals" in the particular regions (lumbosacral, dorsum hand,
dorsum foot).
During this developmental period, immature melanocytes also tended
20 Zl\lMi:iiM.\N.\ AMI HK(Ki;H
to becuint' adluTcnl Ui small Ijluud vessels. Whole chains clearly outlined
the course of caijillaries. The cells often had the ajipeaiance of silver-
impregnated "pericytes." Figure 12 ]ioi trays such an arrangement in the
dermis of the scalp. Migratory melanocytes may also follow the course of
nerves in the dermis. Such migration, however, seems to be incidental
rather than essential. Similar observations by Ehrmann may have induced
him to bclicNc lliat melanoblasts were derived from the adventitia of blood
vessels.
Sixtli Muidlt to Birth. The mateiial for this period consisted of three
specimens of the twenty-sixth and twenty-eighth weeks and of four full-
term fetuses, ill llic full-term fetuses the epidermis was sloughed; the
dermis, however, was well pieserved and excellent silver impregnations of
"dermal" melanocytes were obtained.
Characteristic of this late fetal j^eriod is the disappearance of the
melanoblasts and melanocytes from the dermis of most skin areas. Per-
haps the cells merely lose the active enzyme system that is necessary for
the elaboration of melanin. They would then become undetectable with
(lur present methods. They may disappear entirely, though we cannot
be sure.
E(iually characteristic, however, is the fact that "ilermal" melanocytes
remain a constant feature of the scalp, the sacral region, and the dorsum
of the hand and foot. In the last two regions their number varied between
500 and (iOO cells at six months as well as at birth. In the sacral region of
several newborn specimens we counted from 900 to 1,400 "dermal" mela-
nocytes per mm.- Figure 13 shows such cells from the dorsum of the hand
(if a iiewlioru Negro.
V. The Development of Distribution Patterns of Epidermal Melanocytes
During the Fetal Period
It has been known for some years {Zimmermann and Cornbleet, 194S)
that the first epidermal dendritic cells appear early in the third fetal
month. Beckei' and Zinuiieiiuaim (10.").")) had also establisluHl that in the
iiewliorii Xegro theie are approximate]}' l.UOO melaimcNies per mm'" of
epideiinis. But only incomplete information has been available concerning
the i)o])ulation density of such cells at different fetal periods and in various
skin areas.
Previous data wnv based on skin sections in which the mean distance
between 200 consecutively encountered melanocytes hail been measured.
The ])roi)ai)le number of melanocyt(>s jier nun" was then estimated. It is
difficult to identify fuiictidiially immature dendritic cells in skin sections
of earlv fetal months, in trviiig In avoid llial the same cells were counted
MELAXOBLASTS AXD MELANOCYTES IX FETAL XEGRO SKIX 21
ill adjacent sections, we had measured only alternate sections. This pre-
caution led to cell counts that were too low. Our more reliable, present
method calls for correction of the figures published in our 19.55 study.
Forty-three fetuses of our material had been aborted between the
tenth and twentieth weeks of development. This developmental period
proved to be an important one for the differentiation of epidermal mela-
nocytes and the establishment of their regular distribution pattern. No
intraepidermal dendritic cells could be seen before the tenth week (fetuses
with a C.R.L. of 40 mm or less). In the twentieth week, however (C.R.L.
of over IBO mm) the population density of epidermal melanocytes in most
skin areas already resembled the conditions at birth (800-1,100 per mnr).
The following is an account of the development of distribution patterns of
dendritic cells until birth.
Tenth Week. The earliest epidermal melanocytes were identified in
the interscapular region of a fetus measuring 34 mm in crown-rump length.
Its age was calculated at 2.7 lunar months or 10 3/7 weeks. Widely scat-
tered dendritic cells were observed, some with fairly long, branched proc-
esses. Figure 14 shows one of these earliest intraepidermal cells. Three
dendritic processes extend through a total distance equal to about ten
ordinary epidermal cells. Argentaffin granules of premelanin became vis-
ible with Masson's reduced silver technique. They could not be seen in
unstained preparations nor after treatment with ordinary silver nitrate
(Bizzozzero). Fully elaborated melanin, therefore, was not present. The
dermis contained numerous round melanoblasts, e.g., in the sacral region.
Counts of the sporadically appearing, earliest melanocytes of this stage
would be meaningless.
Eleventh Week. Scattered intraepidermal melanocytes were observed
in various cutaneous areas of eight fetuses of this period. Their crown-
rump length varied between 37 mm and 55 mm. Scalp, cheek, and nape
revealed dendritic cells in most of these specimens, although never in high
numbers. A few cells were also identified in the interscapular, para-
umbilical, and gluteal regions. Even the epidermis of the delicate limbs,
on the dorsum of the arm and the posterior region of the thigh, contained
a few young dendritic cells. In most areas there were fewer than ten early
melanocj'tes per mm'. Cell counts were not reliable. The youngest forms
were fusiform with slender, drawn-out cell bodies and two dendritic proc-
esses. Some were triangular, with indications of three processes, or even
with secondary branches. Some of the processes measured 50 p. in length.
The total length of the fusiform cells usually ranged between 50 and 75 n.
The distal limb segments were still devoid of dendritic cells.
TiceJfth Week. Epidermal melanocytes now appeared also in palm
and sole. The distribution of early dendritic cells was regular enough to
22 ZIMMKUMANN AM) HKCKKK
allow coll (tiiiiits ill five of the seven available fetuses of this period. Their
ciowii-niiiii) Iciifitlis \aiii'(l Ix'twecii .">() and ()7 imn. Tlip cell coiiiifs ranged
from •'iO to L'(M). In two specimens, however, the population density of
youufi dendritic cells already was remarkably high, e.g.. 576 cells per mm'
in the nape and 738 cells per mnr in the sole of one. (503 cells in scalp and
513 in the palm of the other specimen. Since the distribution pattern was
uneven, tiiese counts convey at least an impression of the rapid migration
of melanocytes into specific epidermal areas. Transitional cell forms be-
tween young dendritic cells and ordinary ejiithelial cells were not seen.
Each of the 20 .skin areas contained .some dendritic cells at this early age.
Their cytodif!'erentiation occurred rapidly. Figures 15 and 16 represent
early cell forms and distribution patterns of epidermal melanocytes of a
twelfth week fetus. The fusiform cell bodies of the sole, showing long
primary dendritic processes, were characteristic of young dendritics in
all cutaneous areas. In the sole they had just appeared at this age; in
the nape (figure 16) they had already attained a higher degree of
differentiation.
Figures 17 and IS re))resent cell stages encountered in full-tiiickness
skin spreads of two fetuses obtained from the Carnegie Institution of
Washington. Depaitment of Embryology (Baltimore). Their respective
ages hail been determined in that laboratory as 12 weeks (Carnegie speci-
men Xo. 9014) and 12 3/7 weeks (Carnegie specimen No. 8613). Figure
17 shows highly differentiated melanocytes in the epidermis of the scalp
and figure 18 of the anterior region of the leg. The surface views of these
spreads show the nuclei of ordinary epidermal cells and permit a com-
parison with the length of the dendritic processes. The dendritic cells
reveal accumulations of argentaffin granules to the tips of their processes.
Figures li) and 20 portray conditions of melanocyte differentiation in
the najie and in the dorsum of the arm. The cells are of a uniform type
in the formei- liut varialile in the latter region. The fusiform, slender cell
forms prol)al>!y had recently "aniveii" in the epidermis on the dorsum
of the arm.
A relative "timetable of arrival" of ejiidermal melanocytes in differ-
ent skin areas of an individual is siu)wn liy cell counts. During the twelfth
week they were consistently higher in the palm than in the .«ole. e.g..
351:72; 234:63 per nun-'.
Thirteeiilh mid Fourteenth W'icLs. A markeil increase in tlie numl)er
of epidermal nielanoc^'tes chaiacterized lliis jiiTind. In the palm api^eared
the first indications of rete ridges (ejiidermal crests).
We studied six fetus(>s ranging from (it) to 75 nun C.H.L. in the
thirteenth week and from S5 to 133 nun in the fourteenth week.
Tiie distrii)Ution of Nounu dendritic cells was more imiform and cell
MELAXOBLASTS AND .MELANOCYTES IN FETAL NEGRO SKIX 23
counts became reliable. Cell distribution remained irregular in only a few
skin areas. The orientation of melanocytes often indicated a directional
flow or migration ( figuie 20). Population densities of tlie dendritic cells
varied, both regionally and individually, between 300 and 1,000 per mnr.
In the scalp, cheek, dorsal trunk regions and dorsum of forearm the counts
were between 800 and 1,000 cells. Great waves of rapidly differentiating
dendritic cells thus appear to arrive at their destination during a short
period of only two or three weeks. Their population density ([uickly ap-
proached that of the newborn. Since nuclear events in epidermal mela-
nocytes cannot be observed after sih'cr impregnations, we are unable to
say whether the increase in cells was due, in part, to mitotic divisions.
Figure 21 illustrates the distribution pattern in the scalp of a Negro
fetus of 131/2 weeks (75 mm C.R.L.). We counted 936 dendritic cells per
mm- of that area.
The distrilnition features in palm and sole were of special interest.
When they first appear the melanocytes are fairly evenly scattered
throughout the epidermis of palm and sole. Figure 23 shows that pattern
in the palm at 13 weeks. It changed rapidly with the differentation of
rete ridges (epidermal cristae) between the thirteenth and seventeenth
weeks. This occurs first in the palm. Figure 24 shows the dendritic cells
located almost exclusively on the rete ridges. Palm and sole contain some
melanocytes until birth and even to adult life. Their potentiality to form
melanin, however, appears more inhibited than elsewhere in the body.
Fifteenth and Sixteenth Weeks (end of fourth month). Our material
consisted of three fetuses of the fifteenth week (95 to 105 mm C.R.L.) and
of four fetuses of the sixteenth week (107 to 117 mm C.R.L.). In general,
the distribution patterns of the dendritic cells were regular and uniform
in all skin areas, except in the palm. Cell counts were higher than in
preceding periods. This reflects a continued infiltration of the epidermis
by melanocytes from the dermis. At 15 weeks the population density of
dendritic cells varied between 600 and 1,200 per mm-. Figure 22 shows a
characteristic distribution pattern of epidermal melanocytes in the inter-
scapular region at that time.
In unstained spreads of scalp specimens of the fifteenth and sixteenth
weeks, the dendritic melanocytes were faintly visible. The degree of their
differentiation corresponded to that shown by impregnations witli am-
moniacal silver nitrate. Unstained dendritic cells were also identified at
the palpebral fusion plate. Impregnations by Bizzozzero's method proved
that they contained fully elaborated melanin. We had previously shown
that melanin is first formed in melanocytes of certain head regions early
in the fourth month.
The rete ridges in the jialmar epidermis become well established during
24 ZIMMKUMANN AM) MIOCKKU
the fifteenth week. The previously scattered luehiiiocytes now were located
on the ridges and their nunil)er decreased to l.")()-20() cells per mm-". This
may he due lo a loss of staiiiahilily or to actual disappearance of the
dendritic cells. Duiiiiii ihc fit'tccntli week tlie sole usually has no rete
ridges as yet.
During the sixteenth week, cell counts of epidermal melanocytes varied
between 700 and 1,200 per nun'. The palmar and plantar areas were again
the excei)tions. In the palm all tlie dendritic cells were on the rete ridges
and nunil)cr(Hi between loO and 2.")() cells per luni". In the sole, instead,
the epidermal cristac were just bciiinning to form and tiie number of
the evenly scatter('(| melanocytes remained lelatively high: 37.) to 450
per mm-.
Sev€iit(ciitli and Eiyhtcenth H'crA*-. There were no important changes
in this developmental period. Specimens of the seventeenth week meas-
ured between 125 and 129 nun in C.R.L. and lietween 130 and 137 mm
C.R.L. in the eighteenth week.
In general, the po]nilation density of melanocytes was similar to that
at the end of the fourth month of development (600-1,200 cells per mm").
There were two exceptions. Counts in skin spreads of a 130 mm fetus
were consistently low (300-700 cells). There were some signs of relatively
poor preservation and the counts may not be reliable. I'nusually high cell
counts were obtained in preparations from a fetus of 17 weeks; manj' ex-
ceeded 1,000 per mm-'. I'ixation and silver impregnation were good. We
ascribe such high counts of dendritic cells to individual variation, which
will be discussed in a subsecjuent section.
During the seventeenth and eighteenth weeks, the development of
rete ridges in palm and sole had further ])rogressed and constituted very
characteristic features. The melanocytes were locatetl exclusively on the
ridges. Theii' number was consistently higher in the .st)le than in the palm.
Cell counts in both areas were higher in specimens of the seventeenth than
in the eighteenth week. In the palm the average number per mnr de-
creased from about 200 to 100. in the sole from al)out 400 to 175 cells.
This may l)c due to a loss in functional activity with resulting unstain-
altility, or it may mean an actual decrease in the numlier of melanocytes.
Figure 24 shows the arrangement t)f the melanocytes on the epitiermal
ridges in the palm of a Negro fetus of 17 weeks. There were 126 dendritic
cells per mm' (compare with figures 23 and 26).
X'nu'Ueiith and 'rwcnlicth Weeks (end of fifth month). Split-.^kin
])re])arations were ol)tained from five fetuses l)elonging to the nineteenth
week of development (145 to l.")!) mm C.R.L.) and of two fetuses of the
twentieth week (165 nun C.R.L.). Cell counts fiom one fetus of the
twenty-first week were included in this jteriod.
MELAXOBLASTS AXD MELANOCYTES IX FETAL XEGRO .SKIX 25
The population densities of epidennal melanocytes varied between 500
and 1,000 cells per mm'-. In one fetus it ranged from 700 to 1,100 and in
another between 800 and 1.400,
The impression gained from an analysis of cell counts in the seven-
teenth and eighteenth weeks was sustained: the great influx of melano-
cytes into the epidermis had occurred before the end of the fourth month.
There was now a distinct slowing of that process, A degree of stabilization
appeared to be attained, although various cutaneous areas still contained
melanocytes in the dermis, Barry ( 1953 ) had noticed it but nothing is
known of the causes. We can merely state that the period of penetration
of the epidermis by melanocytes is relatively short and occurs essentially
before the midpoint of pregnancy.
Figure 25 shows a typical distribution pattern of epidermal melano-
cytes in the anterior abdominal wall at 19 weeks. The population density
was 873 cells per mm'-.
The gradual decrease in the number of melanocytes on the epidermal
cristae of the sole is shown by the following figures: 531 per nmi" at 17
weeks, 360 at 19. and 270 at 20 weeks.
Twenty-Sixth Week. We were able to obtain skin specimens of various
body regions from two fetuses of 6.6 months. The counts of epidermal
melanocytes from 10 cutaneous areas of one fetus (220 mm C.R.L.) varied
between 700 and 1.100 per mm-. In the sole there were only 81 dendritic
cells per mnr, all located on the rete ridges. In the other fetus of this
period (225 mm C.R.L. ), the cell counts from seven cutaneous areas ranged
between 1.000 and 1,485.
These counts are in good accord with our findings from the end of the
fifth fetal month and with those in the newborn. They indicate that no
additional wave of melanocyte "arrivals" occurred during the second half
of the fetal period. Scattered melanocytes from the dermis may neverthe-
less enter the epidermis in small numbers.
Full-Term and Negro Injants oj the Xeonatal Period. The epidermis
had been sloughed off in skin specimens of four full-term fetuses. In the
palm of one specimen we were able to count 288 melanocytes per mm".
Figure 26 shows their arrangement on epidermal ridges, between openings
of the sweat pores. In the sole of another specimen there were 171 den-
dritic cells per mm-. Although unsatisfactory for an over-all study of pig-
ment cells in the epidermis, the four fetuses nevertheless revealed great
numbers of melanocytes in the dermis of special areas. Reference to those
findings was made in another section.
Reliable counts of melanocytes in the newborn had been previously
reported from the abdominal wall (Becker and Zimmermann, 1955). Epi-
dermal spreads had been obtained by trypsin digestion. They were fixed
2() ZIMMKHMANX AM) HKCKKH
ill 2 i)cr cent tnriiialiii tnr t'oiii' Ikiius ami llicii iiiciibatecl in a 1 per cent
(lojja solution for three hours. Counts ranged between 083 and 1.152 dopa-
positive denthitic cells per nini". In a ten-day-old Xegro infant, the dopa-
treated ejiiderinis contained between ()17 and S14 melanocytes per iiinr of
abiloiiiiiial surface area.
VI. Regional Differences in the Frequency Distribution of Epidermal
Melanocytes in the Negro Fetus
A total of 412 field counts of ejMdernial melanocytes were made. Only
such preparations were used in which the melanocytes were well impreg-
iialed witii rerjuced silver nitrate. There were differences in the intensity
witii which iii(H\iihial cells took up the silver. Since all preparations were
made with ceiual care, our numerical data are considered adeiiuate for an
estimate of regional tlifferences in population densities. They represent a
first attempt at determining the time of arrival of fetal melanocytes in
various body regions. Wherever feasible 20 cutaneous areas of each fetus
were studied.
Figure 1 graphically illustrates tlie pooled data for two developmental
periods: (a) the twelfth week, when the epidermis is being invaded by
melanocytes in numbers, and (b) a longer interval from the thirteenth to
the twenty-first weeks (end of fifth fetal month). During that time occurs
a stabilization of the cell counts. ( 'onsec|uently. the population density of
epideinial melanocytes leinaiued relatively constant diu'ing the second half
of pregnancy.
During the twelfth week, cell counts in ilifl'erent regions varied from
about 20 to 400 dendritic cells per mnr (see Table 1). A total of 50
counts were made. Tlie highest counts were obtainetl from the scalp,
cheek, nape, and interscapular regions. The counts for various cutaneous
aieas of the upper limb were higher than tho.^e of the lower limb.
.Surprisingly high numbers of dendritic cells were obtained for palm
(351/mnr) and sole (243/nmr).
Since the head region of an embryo and young fetus grows earlier and
faster, and the anterior limb differentiates ahead of the jiosterior limb, the
population densities of nielaiiocytes appear to conform with the general
se<iuence in cephalo-caudal growth. .\\\ anteio-jiosterior gradient in the
distril)ution density of early melanocytes was clearly indicateii.
Relatively high counts in the jialm and sole may be due to an accu-
niiilalioii (if iiiigraloiy niclaiioblasts in distahnost ar(>as. Tlieir in\-asion
of tiie ei)ideiniis and differentiation into ileiidritic c(-lls may occur at a
high rate. The iiopiilation density in tiie palm and sole increased liuriiig
the thirteenth week but decreased tliereafter. .V dorso-ventral difference in
the iiiiiiilier o\' iiielaiiocN'les was iml e\id('ii1 in the Iwcltth week.
MELAXOBLASTS AND MELANOCYTES IN FETAL NEGRO SKIN 27
COMBINED AVERAGE COUNTS OF DENDRITIC MELANOCYTES PER MM
HOG
1000-
900-
800-
700-
600-
500-
400-
300-
200
100
Heod ond Trunk
13th -21 St fetal week
(TOTAL COUNTS^ 270)
Upper Limb
I2th fetal week
Lower Limb
J„
Figure 1. Population densities of ejiidermal melanocytes in various loody regions.
The lower curve shows average counts of melanocytes per mm- during the
twelfth fetal week. The upper curve represents pooled frequencies between the
thirteenth and twenty-first fetal weeks. Tlie number of melanocytes in palm and
sole dro]3s sharply after tlie thirteenth week.
The top portion of the graphs in figure 1 shows the average frequency
distribution of epidermal melanocytes in 20 areas of fetuses between the
thirteenth and twenty-first week. It is based on 270 field counts of den-
dritic cells.
The data were first plotted separately for the thirteenth week (25
counts), for the fourteenth to sixteenth week (149 counts) and for the
nineteenth to twenty-first week (96 counts). Since there was an overlap of
population densities in corresponding areas we pooled the entire group of
data. The graph portrays the main trend of regional cell frequencies dur-
ing the fourth and fifth fetal months.
In fetuses of the thirteenth week the counts of epidermal melanocytes
were significantly lower for the lower limb as compared with those of the
upper limb. This agrees with the slower or delayed rate of development
of the hind limb. In the following few weeks that difference in population
densities became rapidly erased.
Between the thirteenth and twenty-first weeks the average counts of
28 ZIMMKH.MANN AM) HKIKKK
luclaiiocytt's ltd' all culaiicuus icfiitjiis xaiied ht'twciMi <).")() anil it.jU iimi".
The greatest influx of epklennal inelanoeytes into any of these areas
had occurred Ix'twccn the twclt'ih ami fouitccntli weeks of intrauterine
(levelopnicnt.
The most characteristic features were the gradual disapiiearance of a
cephalo-cauilal gradient, and the clear emergence of a dorso-ventral gra-
dient ill jxipulatidn densities, tor the trunk. ui)i)er and lower limb.
Till' cdunts (if melanocytes ilid luit icmain significantly higher in the
(■(>plialic portions of the fetus. High counts were obtained from the sacral
region. The number of dendritic cells in dorsal areas of both limbs was
also approaching the previously higher counts in the head region.
The ])ool('d axcragc counts fiDin the doisal areas of trunk and limbs
(littered from those of corresjionding ventral areas. They were significantly
higher in the sacral area, dorsum of arm, forearm and hand, posterior
region of thigh, and in the calf, than those of the paraumbilical and pec-
toral regions, or of anterior areas of arm, forearm, thigh, and leg. This is
clear evidence of a dorso-vential giadient in tlie pojudation densities of
melanocytes at that fetal period.
The high counts in the sacral legion (average: !)47/mm-) are of
special interest since that area corresponds to the caudal end of the neural
tube and of the neural crests. Great numbers of pigmentary precursor
cells accumulate in the dermis of that area. They constitute tlie substrate
foi' the so-call('(i .Mongolian s])ot. .Many of tiies(> cells may later penetrate
the epideiniis and thus lead to a liigh po])ulation density of dendritic cells
in the sacral area. All the epidermal melanocytes of the lower limb, inci-
dentally, must also be derived from the dermal pool of melanoblasts in
that area.
Sjiecial features prevailed in the epidermis of palm and sole of the
fourth and fiftli months. The first epideiinal cristae or rete I'idges appear
ill the i)alni about two weeks earlier than in the sole. They were easily
identified in both areas during the eighteenth week. None were present
elsewhere in the skin of that period. During the formation of rete ridges,
the counts of palmai' and jilantar melanocytes dropped sharply. Their dis-
li'iliutioii ])att('in changed from one of cNciily scattered cells to one of
restricte(| alignments on the rete ridges. There remained only rare den-
dritic cells Ijetween the epidermal crests. This decrease is perhaps more
simulated than real; if the enzymatic activity necessary for melanin pro-
duction lu'canie inhibited, the cells would not be revealed by our impreg-
nation techniciue. Inliiliition of tyrosinase activity by SH groups has been
j)roved to occur in adult wiiite skin. In tlu' jialm and sole of Negroes it
begins ])erhaps as early as the fourth fetal month.
MELANOBLASTS A\D MELANOCYTES IX FETAL NEdRO SKIN 29
VII. Individual Variability in the Population Densities
of Melanocytes During the Fetal Period
The poohng of cell counts tluough a relatively extensive develop-
mental period, as done in the foregoing section, has the advantage of
diminishing the effects of minor technical errors and of bringing out the
major trends of conditions under study. Such a procedure has the dis-
advantage, however, of erasing iiuUvidual differences in the population
density of melanocytes. The "average truth" is not the whole ti'uth in
these matters, any more than in many others.
A possible source of error resides also in relationship between the
degree of differentiation of a specimen and its age as determined by exter-
nal body dimensions. Streeter showed in his "horizons of human develop-
ment" that an identical degree of interior differentiation may be attained
in embryos of different external dimensions. During fetal development
that discrepancy probably is less significant. Our age determinations ac-
cording to Scammon and Calkin's formulae are valid for comjiarisons, but
they do not necessarily reflect absolute age. Any errors in this respect
probably would affect age determinations by not more than one week.
Already in the twelfth week we encountered one specimen in which
the average count of melanocytes per mm- was much higher (540) than
the entire range for other individuals of that period (50-200). A similar
difference between specimens of practically identical crown-rump lengths
was noted at 15 weeks. The discrepancy in cell counts was especially
apparent in corresponding skin areas of the lower limbs. Again in the
eighteenth week we obtained range variations in cell counts from 300-700
in one individual and from 600-1,000 in another. The range of variability
generally was between 500-1,000 melanocytes/mm- in specimens of the
nineteenth week. In one individual of the twentieth week the counts
varied from 700 to 1.100 cells and in another fetus of the twenty-first week
from 500 to 900. In two fetuses of the twenty-sixth week the cell counts
(disregarding palm and sole) were between 675 and 1,100 per nmr in one
specimen, and between 1,000 and 1,485 in the other. Counts in skin speci-
mens of newborn Negroes averaged 1,035 melanocytes per nun-. Unfor-
tunately we were not able to obtain material from many cutaneous areas of
that age.
The above-reported individual differences might be considered char-
acteristic only for fetal stages. Our data do not extend far into postnatal
life and we cannot say whether such differences persist. It is well estab-
lished, however, that they exist both in white and Negro adults.
30 ZIMMKHMANN AND HKCKKli
VIII. Discussion
111 the ])ast, cutaiioous pifiincnt cells and their precursor stapes have
been known !)>■ \'ari()us names. To the jn'oponents of a theory that preceded
Hloch's. tlu\\- were tixcd conncri ivc tissue cells that had penetrated the
epidermis. Accordinji to Mlirmann. pigiiieiit-i)roducinf;- cells were "melaiio-
blasts," located in the dermis or in the epidermis and presumed to be
derived from embryonic mesoderm. But Bloch insisted that they were
of ectodermal origin, arising in the basal layer of the e]Mdennis. Biologists
generally jireferred the terms chromatophores or melano])liores. evidently
disregarding the fundamental difference between carrying melanin and
actively producing it.
Adachi, with other distinguished authors of his time, believed that
"epidermal chromatophores'" were an illusion. ^Melanin of the mammalian
and human epidermis was not contained in cells at all but in intercellular
spaces. Others, i)articularly P>ench authors, designated the epidermal
pigmentary elements as "Langerhans cells." Paul Langerhans (1868) had
discovered stellate cell forms in adult white skin by means of gold imjireg-
iiations. He believed them to be nerve cells and did not associate them
with pigment ]iroduction. Later these elements were considered either as
artifacts oi- identical with the true ]5igment-prii(lucing dendritic cells of the
epidermis.
According to Masson (1!)48), "Langerhans cells" represent effete
melanocytes that have lost their ability to produce melanin and are
ajiproaching desciuamation. Billingham and Medawar (1953) emphasized
tliat only "high level" branched cells in the epidermis are identical with
"Langerhans cells" and that the latter did not occur in the basal layer. In
adult heavily pigmented skin the high level dendritic cells never are ilojxi
positive. They have either lost or discharged their pigment and are an
exhausted type of cell.
In fetal Negro skin no distinction can be made between branchetl cells
in superficial or deep layers of the epiilermis. All are functionally active as
pigment producers. Consequently, there are no "Langerhans cells" in
fetal Negro skin. This confirms the interpretation that in adult skin they
represent "spent" melanocytes. Hence we have not usetl the term.
Masson (1948) designated the pigment-producing elements of the
epidermis as "clear cells" or "cellules claires." Tlie term staiuls for cell
bodies (perikarya) that lie mostly in the basal layer and usually show
some pigmentary activity. They were even described as lymphocytes
penetrating the epidermis. Billingham and Medawar (19.13) ha\e drawn
attention to that erroneous interpretation.
liloch (1917) and Beckei- (1927) called the dopa-]iosili\e biancluMl
cells I)( iiilrlh iizi ll( II nv (lench'ilic cells. These leiins were ])arl icularly
MELAXOBLASTS AND MELANOCYTES IN FETAL NEGRO SKIN 31
useful in the earlier days of pigment research when neither their origin nor
true nature was known. "Dendritic cell" now is a widely used term
(Billingham. 1948). It is descriptive, noncommittal, and applicable to all
epidermal stellate cells, whether they have pigmentary activity or not.
In this bewililering array of more or less synonymous terms — which
naturally has led to confusion — the best name for pigment cells is "mel-
anocytes." We have applied it both to immature, branched forms of
pigment-producing cells in the fetal dermis and to the highly differentiated
dendritic cells of the epidermis. Pigmentary as well as inhibited dendritic
cells are melanocytes. Alelanoblasts, instead, are early embryonic cell
types, round or ovoid, and re\'eal the first signs of their j^igment-producing
potentiality.
During the past 75 years a bi'oad concept has evolved relative to
melanocytes in the human dermis. At first recognized only in the sacral
region of Japanese fetuses and children, then in the dermis of certain
primates, the same cells were subsequently described in the sacral region
of whites, in blue nevi, and finally in the dermis of several body regions of
early fetuses of the yellow race. We have shown that during early fetal
months, immature melanocytes are present in the dermis of all body
regions of Negro fetuses. j\Ieasurements of the characteristic cells corre-
spond closely to those given by previous authors for the so-called Mon-
golian cells. Barry (1953) postulated that "dermal melanocytes are the
forerunners of dendritic Langerhans cells." He fuither emphasized that
the pigment cells of the Mongolian spot, of the blue nevi, of the hair
matrix, and the dendritic cells of the epideimis are cytogenetically related
to each other. In general, we agree with this conclusion.
We have found that in later fetal life and at biith, "dermal" melano-
cytes remain identifiable only in certain skin areas: in the scalp, the sacral
region, and the dorsum of the hand and foot. Temporarily, all cutaneous
areas of Xegro fetuses contain some pignient-j^roducing cells in the dermis.
These melanocytes appear in every way homologous to the permanently
present pigment cells in the dermis of certain anthropoid apes and
monkeys.
Their gradual disappearance from the dermis of most skin areas needs
further investigation. The present methods did not permit us to establish
whether many of the melanocytes degenerate or whether their enzyme
system merely becomes inactive. Possibly some of the cells ascend to the
dermo-epidermal junction and mature into highly branched epidermal
melanocytes.
Figure 2 shows our interpretation of the paths followed by melano-
blasts and melanocytes. Since melanoblasts were not seen before the tenth
week of menstrual age, parts A and B of the diagram remain hypothetical.
32 ZIMMKHMANX AND HKCKKIi
yt
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eos (Mong>
Blue Nevi
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MELANOBLASTS AXD MELANOCYTES IN FETAL NEGRO SKIN 33
They are based on experimental evidence in mammals (Rawles, 1947).
The remainder of the schematic representation is self-explanatory. The
cell types shown were dra\\-n from high po^^•er observations. The evidence
for our views, of course, is mainly circumstantial since actual cell migra-
tions and transformations cannot be observed in fixed tissues. This is a
corollary of a purely morphological or histological approach.
Another question pertains to the multiplication rates of melanoblasts
and melanocytes in their migratory phase. Hu, et al. (1957) have shown
that melanocytes in cultured sheets of Xegro epidermis underwent mitotic
divisions. Billingham and Sparrow (quoted by Billingham and Aledawar,
1953) have proved experimentally that the pigmented epidermis is "a
reproductively self-sufficient system." Cell divisions occurred in both ^lal-
pighian cells and melanocytes. Masson (1948) was convinced that "mel-
anoblasts" multiply within the basal layer of the human epidermis. He
noticed features suggesting amitotic division and only once recognized a
mitotic figure. Cell divisions of epidermal melanocytes have also been
reported by Pinkus (1949) in a wart and by Becker Jr., Fitzpatrick, and
Montgomery (1952).
We were unable to study nuclear events, since the silver impregnations
of premelanin and melanin granules largely obscure them. Many cell
forms of immature melanocytes in the dermis recalled Masson's observa-
tion. The manner of their possible multiplication needs further study. The
numerous round melanoblasts in the scalp in late fetal periods and even
at birth are also puzzling. Both cell forms may represent preparatory
phases of cell division, ratliei' tlian new cells derived from the neural crest
(Weissenfels, 1956). Other staining methods will have to be employed to
obtain a flefinite solution to this problem.
The main data on population densities of melanocytes in various body
regions and at different times of the fetal period are briefly summarized in
the conclusions of the present study.
Previous studies have shown that the average number of epidermal
melanocytes per unit area is not significantly different in adult individuals
of the white and Xegro races. ^Marked numerical differences, however, do
exist between individuals of either race (Szabo, 1954; Staricco and Pinkus,
1957). We found similar individual variations to exist already in the
fetal period of X'egroes. Our data indicate that differences in population
densities of melanocytes, both regional and individual, probably become
established relatively early in fetal life.
34 zi.MMi:i(MA.\.\ AM) ni;(Ki;u
IX. Summary
'I'hiii lioiizoiital sli;uiiiji.s of tVtal Xi'jiiu t^kiii were iinprejiiiated with
ainiiioiiiiical silver iiiliatc. A inodificfl Masson tcchiiiciuo was used and the
split-skin picpaiatioiis wcic iiKnintcd as spreads. The material consisted
of 1()() fetuses, varyiiifi in ajje from the seventh week to birth. Twenty
selected skin areas were studies in each fetus. Over 500 field counts of
pigment cells in the dermis and epidermis were made.
The earliest precursor stages were idcntifiiMl in the dermis of the tenth
week of development. They appeared as round nielanoblasts containing
the first argentaffin granules. Still younger forms of that embryonic cell
type could iiot be distinguished from mesenchymal cells. A gap of a few
weeks remains between the development of the neural crest in man and
the earliest identification of its presumptive pigmentary elements. Experi-
mental evidence in mammals has closed that gap (Rawles).
Between the tenth and twelfth weeks, nielanoblasts occur in the dermis
in increasing numbers; the epidermis then contains only scattered den-
dritic cells. Mclanoblasts dift'erentiate into spindle-shaped, immature
melanocytes with two or three short, stubby processes and increasing
amounts of pi'cmelanin granules. They were identified in the dermis of all
body regions, in groujjs or "swarms." Cell counts were highh' A^ariable.
Immature melanocytes were recognized in unstained preparations as
early as the fifteenth week. Average counts of 300 to 400 per mnr' were
obtained in the thirteenth and fourteenth weeks, of 500 to 700 in the
fifteenth and sixteenth weeks, and over 1,000 in the sacral region from the
seventeenth to the twentieth week. From the sixth month to birth,
"dermal" melanocytes were found only in the scalp, sacral area, and in the
dorsum of hand and foot.
The first few epidermal melanocytes were observed in the eleventh
week. Their numbers per mm- increased sharply between the twelfth and
fourteenth weeks, indicating a ])eriod of rapid infiux. No transitional
forms of ordinary basal cells were seen. At first fusifoi'm with long pri-
mary ])rocesses, the epidermal melanocytes rapidlj' diti'erentiated into large
dendritic cells. As early as the twelfth week they appeared in most body
regions, including palm and sole. Uniform distribution patterns and high
poiiulation densities became established before the end of the fourth
month (SOO to 1,000 melanocytes jier nun-'). Their number decreasetl in
palm and sole with the deNeluiiiiicnt of rele ridg(>s (sixteiMith to nine-
teenth weeks), but some melanocytes remain on the ridges until i)irth.
Population densities of ejiideiinal melanocytes remained fairly stabilized
after the fifth fetal nmnlh. in the newborn Negro there were approxi-
mately' l.Oiv") dopa-])osil i\e dendritic cells per iinn-'.
Regional and in(li\idual dilTerences in poimlation densities of fetal
MELANOBLASTS AND MELANOCYTES IN FETAL NEGRO SKIN 35
melanocytes were observed. An early cephalo-caudal gradient later dis-
appeareil. It probably expressed different times of "arrival" of epidermal
melanocytes in various body regions. The earliest fully elaborated melanin
first appeared in certain head regions (third month). Gradually a dorso-
ventral gradient in population density of epitlermal melanocytes emerged
and remained until birth, especially in the trunk and upper limb regions.
Individual differences in jiopulation densities were noticed as early
as the fourth fetal month.
30 ZIMMKIIMANN AM) Hi;( KKK
X. Literature Cited
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Mdipli. u. .\ntlii(i|i. li: 1-131.
Baklz. K. 1885. Die krupcrliclicn Eisicnscliafteii (Icr .lapaiK r. Mittcilp. DcTitst-li.
(it'.-:i'li.-^cli. f. Natur-u. \'olkc'rkun(it' O^-tasion.s. 4:40.
Bakuv. .1. 1953. Uccliirclics sur I'originc dcs cellules de Langerhans de repiderme.
Biol. med. 4,-': 293 -31.",.
Bkckkr, S. W. 1927. Mthiiiin iiiiiiiuiitaiiini. A .-^y.-tiiuatic .-^tiuly of tiie ]iigincnt
(if tlic iiuiiiaii skill and upper iiiucous iiieiiibranci?. with P]iccial consideration
of pigmented dendritic cclL-^. Arch, of Derinat. and Syi)h. J'j:259-290.
Bkckeu, S. W., Jr., Th. B. Fitzp.\trick. and H. Mo.nt(;omery. 1952. Human
inelanogcnesis — Cytology and histology of jiigment cells. A.M. A. Arch, of
Dermat. and Sypli. 6V;:511-523.
Becker, S. W., Jr., and A. A. Zi.m.mkk.maxn. 1955. Further stuilies on melano-
cytes and mclanogcncsis in the human fetus and newborn. J. Invest. Dermat.
;85: 103-112.
BiLLiNCHAM, R. E. 1948. Dendritic cells. J. Anat, ,9.^:93-109.
. 1949. Dendritic cells in jiigniented human skin. J. Anat. 55:109-115.
and P. B. AIedawar. 1953. A study of the branched cells of the mam-
malian epidermis with special references to the fate of their division products.
Phil. Trans. Roy. Soc. London, Scries B, ^57:151-171.
Bloch, B. 1901. Preuvcs atavi(iucs de la (ransfonuatiim des races. Bull. Soc.
Anthrop. Paris, V Series. ;?:618.
. 1917. Das Problem der Pigiuenlliildung in der Haul. Arch. f. Dermat. u.
Sypli. i,^4: 129-208.
. 1921. Leber die Entwicklung des Haut — und Haarpigmcntcs beim
menschlichcn Embryo und ueljer das Erloeschcn der Pignientbildung im er-
grauenden Haar. Arch. f. Dt'rniat. u. Syph. 755:77-108.
Dannkkl, R., and G. ("i.ekk.maxx. 1954. Die Einwanderunsi der Pigmcntzcllcn
in die Haut und die Haarc bei Nagetieren. Biol. Zentralbl. ?.>:414-428.
EiiHM.WN, S. 1885. Lntersucliungen ut'bcr die Physiologic und Pathologic des
Hautpignientcs. .\rch. f. Dermat. u. Syph. 77:507-532.
Ei, Bahhawv. .v. ,\. 1922. Leber den Mongolenfieck \n'\ iMiropilirii. Arch. f.
DeniKil. u. Sypii. 74/: 171-192.
EsciiiiiciiT, D. F. 1849. Zool.-aiiat.-jiliysiol. Liitersiuliungen iibi'i- die nor-
disciieii Walltiere. 7:70. Ti. \'oss, Leipzig.
(luiM.M, F. 1895. Beitriigc zum Studiiiiii des Pigiiieiits. Dermat. Zeitschr. i:328-
343.
Iliii'Ki.\s I'ux. .\l. 1949. .\iial\>is of smiie phases of iiirlaiiolil.-ist migration in the
l)arred I'lyiiioulli Rock embryos. Physiol. Zool. ,^,^:l-22.
Ilr. I''., R. Iv SxAiutTo. II. PiXKi s. and R. P. FosNAiiiii. 1957. Human iiicl.ano-
i\tes in tissue culture. .1. liu'est. Dermat. As": 15-32.
IsiiiKAw.v, X. 1924. Leiier den sogcuannten Mtmgolenfleck l)ei Japanisclicn
Foett'ii. Folia anat. japouica, i:l-4.
Pro, M. 1953. Stiulies on Melanin. Tohoku .1. I'.xper. .Med. •'i i, Suppl. 1.
MELANOBLASTS AND MELANOCYTES IX FETAL XEGRO SKIX 3/
. 1957. Studies on Melanin. Report II. Tuhokii J. Exper. Med. 65. Siippl. V.
K.\TO, T. 1905. Anatomisch-liiftologische Stiidien iiber die sog. Kinderflcckc.
Mitteilg. :Med. Fakult. Univ. Tokyo tf: 377-396.
Laxgherhaxs. p. 1868. Ueber die Xerven dor Haiit. Virchow's Arch. 44:3'2o-337.
Massox, p. 1948. Pigment cells in man. in 'Biology of ^lelanoma?," Spec. Publ.
X. Y. Acad. Sci., 6:15-51.
^IiESCHER. G. 1922. Die Chromatophorcn in der Haut des Mensclien. Arch. f.
Dennat. u. Sypli. ;o'i :313-425.
MoRisox. Dr. 1889. Beitriige zur Frage von der Pigmentbildung in der Neger-
haut. Monatsheftc f. prakt. Dennat. ,9:485-490.
PixKUS. H. 1949. Mitotic division of lunnan denilritic mclanoblasts. J. Invest.
Dermat. 75:309-311.
Rawles, M. E. 1947. Origin of pigment cells from the neural crest in the mouse
embryo. Physiol. Zool. 20:248-266.
. 1948. Origin of melanophorcs in development of color patterns in verte-
brates. Physiol. Reviews, A'^: 382-408.
ScAMMOx, R. E.. and L. A. Calkins. 1929. The tlevek)i)ment and growth of the
external dimensions of the human body in the fetal period. University of Min-
nesota Press.
St.\ricco, R. J., and H. Pixkis. 1957. (.Quantitative and qualitative data on the
pigment cells of adult human epidermis. J. Invest. Dermat. 25:33-45.
Szabo, G. 1954. The numi)er of melanocytes in lunnan eiiidermis. Biit. Med.
Jour. ;: 1016-1020.
Thomsox, A. 1891. Note on the skin and seal]) of the Negro fetus. J. Anat.
Physiol. i.J: 282-285.
Weissexfels, X. 1956. Licht-phasenkontrast-und elektronenmikniskoiiische Un-
tersuchungen iiber die Entstehung der Propigment-Granuia in Melanoblasten-
kulturen, Zeitschr. f. Zellforsch. 45:60-73.
Zimmer.mann. a. a. 1950. The development of epidermal pigmentation in the
Xegro fetus. Proc. 2nd conf. on biology of norm, and aty])ical pigment ceil
growth. Zoologica. 55:10-12.
. 1954. Die Entwicklung tier Hautfarbe beim Xeger vor der (ieburt.
]Mitteilg. Thurg. Xaturforsch. Gesellsch. (Switzcrlanili 57:33-71.
and Th. Corxbleet. 1948. The develoimient of epidermal |iigmentation
in the Xegro fetus. J. Invest. Dermat. ;;:383-392.
PLATES
Figures 3 to 26 are surface views of eitlicr full-
thickness or split-skin preparations of Negro fetuses.
All except that shown by figure 7 were impregnated
with reduced silver by a modified Masson technique.
Figure 7 represents an unstained preparation. The
magnification varies between 700 and 800 X .
39
PLATE 1
Furt'aiiii. 12 wciks. Full-thickness skin spreads.
Early precursur ^tai^c^: oi nu'lanocytcs in the dermis.
3 Round nu'lanublast, 8-10 /i diauR'tcr.
4 Early iiinnature niclaiidcytt' t'drniing three ])roc-
esscs. 12-16/1.
5 Early fusilorni iiiiniature melanocyte, 20-25 {i.
6 Highly fusiform immature melanocyte 40-50 /i.
40
;SPf;-
'jw^
41
PLATE 2
7 Scalp, 15 weeks. Uiistaiiu'il. Two spindle-shaped,
iiuniaturc melanocytes in the dermis. A small hldud
vessel is coursing between them.
8 Dorsum of foot, 14 weeks. Fusiform melanocytes in
the dennis.
9 Lvuiibdsacral region, 13 weeks. Fusiform and early
stellate forms of iniiiiature melanocytes in the
dermis.
42
43
PLATE 3
10 Dorsum of liaiul, IG weeks. \'ari(ius forms of im-
mature melanocytes.
11 Sacral region (Mongolian spot I, 19 weeks. Round
and fusiform, immature melanocytes.
44
45
PLATE 4
12 Scalp, 18 weeks. AlelaiKR'vtes adjacent to a small
Ijlood vessel.
13 Dorsum of hand, full-term fetus. Round, fusiform
and stellate types of melanocytes in the dermis.
4(1
47
PLATK 5
14 InttTscainilar legion, 10' i; wt'ck.s. Earlici^t oi)iikT-
mal melanocyte witli three ])roeesses. Neighboring
nuclei are of basal epidermal cells.
15 Sole, 12 weeks. Some of tlie earliest melanocytes
appealing in the e])i(leniiis are highly fusiform,
usually showing two long processes.
16 Nape, 12 weeks. Early epidermal melanocytes
with 2-3 dendritic ])rocesses.
48
49
PLATE 6
17 Scalii. 12 weeks. Earliest epiileniial melanocytes in
small groups, irregularly scattered tludugh the epi-
dcnnis. Cell counts are unreliable.
18 Anterior region of leg, 12 weeks. Large, heavily
stained melanocytes appear here and there. The
majority of epidermal melanocytes are smaller and
relatively uniform in size. Neighboring nuclei rep-
resent basal cells.
50
51
PLATE 7
19 Nape, I2Y2 weeks. Tlic uiuqual degree of silver
impregnation of various melanocytes probably re-
flects uncfiual maturity or functional activity.
20 Dorsum of arm, 12 weeks. Fusiform anil stellate,
early dendritic cells.
52
53
PLATE 8
KifliiT distribution i);itl(.Tiis of I'piilfiiiial uiflaiio-
cytos.
21 Scalp, 13^0 weeks. Regular distribution and fairly
even size of ciiidcrmal melanocytes in an intcrfol-
licular field.
22 Interscapular reiiion, 1.5 weeks. The luelanoeytes
appear to be oriented with their long axes ruiniing
parallel to each other.
54
55
PLATE 9
23 Palm, 13' j weeks. Fairly i'\'en ilistrihiitidii of ei>i-
dei'iiial iiiclanoeytes hi'lore retc ritlges (ei)i(lcrnial
crests I are dc^'i'liiiuMJ.
24 Palm, 17 weeks, ^^■itll the development of rcte
ridges the melanocytes come to lie on the crests.
Note the de\elo|)inir sweat pores.
56
■ --^ ,- ■'■■
ik
23
57
PLATE 10
25 Anterior ahildiiiinal wall, 19 wuek?. Rich pattern
of intracpidt'rmal dendritic cells.
26 Palm, newborn. Epidermal melanocytes and sweat
pores are recognizable on the rcte ridges.
58
^
w
'S
--10:
\ .0 y
59
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